Chimeric mouse/chimpanzee liver models: This mouse model is being developed to study HCV virus infection. The basic principle of the model is to induce death of the mouse hepatocytes. Human or chimpanzee hepatocytes can then be transplanted into the mouse liver and will survive due to the growth factors produced by the regenerating mouse liver. This transplanted liver can serve as a substrate for HCV replication. True antibody neutralization could be studied in these mice as well as a number of other aspects of HCV immunology and replication. The original transgenic mice, ALB-uPA, which express the urokinase plasminogen activator gene under control of the albumin promoter crossed with SCID-beige immunodeficient mice can support human liver implants. However, these mice are poor breeders, and the mortality rate for the homozygous ALB-uPA mice is high. We have developed a transgenic mouse that expresses the E. coli nitroreductase gene under the albumin promoter. These mice are healthy until given a nontoxic prodrug, CB1954, which is converted to a toxin by nitroreductase. These mice have now been bred to SCID-bg mice and once a colony is established, they can be tested for their ability to sustain chimpanzee or human liver for HCV propagation. HCV Vaccines: Both CD4+ and CD8+ memory T cells are important in clearance of HCV. We showed that following reinfection of chimpanzees that resolve acute infections the animals, although not protected from reinfection, could rapidly eliminate the second infection with lower levels of viremia and hepatitis. This control was associated with vigorous T cell responses. As a result of these studies we have begun to address the effects of T-cell based vaccines on prevention or clearance of HCV. Dendritic cell based vaccine: A dendritic cell (DC) based vaccine intended for immunotherapy was tested in a carrier chimpanzee. DCs are professional antigen presenting cells and have a high capacity to stimulate T cells specific for particular antigens. This vaccine was designed to induce higher levels of CD4+ T cells specific for the NS3 antigen of HCV. Administration of DCs loaded with the NS3 antigen to the carrier chimpanzee led to an up-regulation of T cells specific for this antigen. However, these responses were insufficient to alter the course of the chronic infection. Therapeutic T cell vaccine: A therapeutic vaccine was tested in a second chronic chimpanzee. This study was designed to augment the T cells responses in the peripheral blood to non-structural proteins of HCV (NS3, NS5A and NS5B) using recombinant vaccinia virus. In this case although the T cell responses were stimulated in the chronic animal there was no effect on viremia and no increase in liver enzyme levels. These results suggested that the vaccines would be more effective if targeted to the liver of the animals. Recent studies have shown that the T cell responses in the peripheral blood do not necessarily reflect intrahepatic responses. Liver Targeted HCV vaccine: The aim of this study is to develop a T cell based vaccine delivered to the liver through the use of modified liposome/DNA complexes and non replicating recombinant adenoviruses expressing non-structural proteins of HCV. HCV Pathogenesis: Functional Effects of In Vivo HCV Mutations: The molecular evolution of monoclonal HCV in chronically infected chimpanzees has identified several newly emerging mutations within the HCV RNA dependent RNA polymerase (NS5B). Some of these mutations have been shown to enhance the activity of the polymerase in vitro, but others have nearly eliminated the in vitro activity. The effects of these mutations on HCV replication using the 1a sub genomic replicon system are currently being studied. Mutations that were seen to occur simultaneously in proteins thought to comprise the HCV replication complex are being introduced together, or alone, in the replicon genome and the growth characteristics of the mutants analyzed. Host Responses Associated with Outcome of Infection: To study determinants of clinical outcome following HCV infection, viral kinetics, immune events and intrahepatic cytokine markers were compared in 10 naive, Patr-diverse chimpanzees infected with clonal HCV. Four of the animals cleared HCV and 6 developed persistent infections. All animals developed similar acute infections with increasing viremia from 1-2 weeks, followed by ALT elevations and seroconversion. An initial biphasic viral increase, a rapid slope (mean doubling time t2=0.5 days) followed by a slower slope after the second week (t2=7.5 days), correlated with increased intrahepatic 2OAS1 mRNA levels. An effective control of virus replication was only observed following increases in intrahepatic IFN-g mRNA, which temporally correlated with ALT elevations. Animals that developed persistent infections had significantly later maximal ALT peaks (p<0.05) and IFN-g peaks than those that cleared the virus resulting in prolonged RNA replication. These data indicate that regardless of outcome chimpanzees generate responses that control HCV replication during the early and late acute phase in a similar manner. This suggests the pathogenesis of HCV infections is subtly controlled in that a more rapid onset of the induced response seems to predict clearance of the virus rather than the presence or absence of such a response.